Natural infection of cattle with an atypical 'HoBi'-like pestivirus - Implications for BVD control and for the safety of biological products

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Natural infection of cattle with an atypical ’HoBi’-like

pestivirus - Implications for BVD control and for the

safety of biological products

Karl Ståhl, Jaruwan Kampa, Stefan Alenius, Annie Persson Wadman, Claudia

Baule, Suneerat Aiumlamai, Sándor Belák

To cite this version:

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Short note

Natural infection of cattle with an atypical

‘HoBi’-like pestivirus – Implications for BVD control

and for the safety of biological products

Karl S



a

*, Jaruwan K



b,c

_{, Stefan A}



c

_{, Annie P}



W



d

, Claudia B



a

, Suneerat A



b

, Sándor B

´

a a_{Joint R&D Division, Departments of Virology, The National Veterinary Institute (SVA)}

& The Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden

b_{Faculty of Veterinary Medicine, Khon Kaen University, Thailand}

c_{Department of Clinical Sciences, The Swedish University of Agricultural Sciences (SLU),}

Uppsala, Sweden

d_{Department of Virology, SVA, Uppsala, Sweden}

(Received 6 October 2006; accepted 8 January 2007)

Abstract – During a study on Bovine Viral Diarrhoea (BVD) epidemiology in Thailand, a pestivirus

was detected in serum from a calf. Comparative nucleotide sequence analysis showed that this virus was closely related to a recently described atypical pestivirus (D32/00_‘HoBi’) that was first iso-lated from a batch of foetal calf serum collected in Brazil. The results from virus neutralisation tests performed on sera collected from cattle in the herd of the infected calf, showed that these cattle had markedly higher antibody titres against the atypical pestivirus ‘HoBi’ than against Bovine Viral Diarrhoea Virus types 1 and 2, or Border Disease Virus. The results also supported, consequently, the results from the molecular analysis, and demonstrated that a ‘HoBi’-like pestivirus had been in-troduced to, and was now circulating in the herd. This study is the first to report a natural infection in cattle with a virus related to this atypical pestivirus, and it suggests that this group of pestiviruses may already be spread in cattle populations. The findings have implications for BVD control and for the biosafety of vaccines and other biological products produced with foetal calf serum. Con-sequently, these atypical pestiviruses should be included in serological assays, and any diagnostic assay aimed at detection of pestiviruses in biological products or animals should be tested for its ability to detect them.

BVDV/ pestivirus / control / biosafety

1. INTRODUCTION

Bovine Viral Diarrhoea Virus (BVDV) is the denomination of a heterogeneous group of viruses in the family

Flaviviri-dae, genus Pestivirus, which are

eco-* Corresponding author:

Karl.Stahl@bvf.slu.se, Karl.Stahl@sva.se

nomically important pathogens worldwide, that primarily infect ruminants [11]. Cur-rently there are two recognised genotypes of BVDV (BVDV-1, and -2) that to-gether with Border Disease Virus (BDV) and Classical Swine Fever Virus (CSFV) constitute the four accepted species of the genus Pestivirus. In addition, a ten-tative fifth species is represented by

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518 K. Ståhl et al.

a virus isolated from a giraﬀe [1]. The pestivirus genome consists of single stranded positive-sense RNA of approx-imately 12.3 kb, with one open reading frame flanked by 5’ and 3’ non-coding re-gions (NCR). Classification of pestiviruses is based on the genetic relatedness be-tween isolates, including genetic similarity with the type virus of the species [6]. Fur-thermore, because pestiviruses are serolog-ically cross-reactive, species demarcation considers antigenic relationships based on binding assays with monoclonal antibodies or cross-neutralisation assays with poly-clonal antisera [1, 5]. Recently, an atypical pestivirus named D32/00_‘HoBi’ was iso-lated from foetal calf serum (FCS) origi-nating from Brazil [13]. Based on genetic and antigenic properties that diﬀered from previously described species within the genus, this virus was proposed as a rep-resentative of a possible novel pestivirus species. Furthermore, the results from in vitro and in vivo experiments suggested ru-minants within the family Bovidae, genus

Bos, as the most probable host species of

this virus. This study is the first to report a natural infection in cattle with a virus re-lated to this atypical pestivirus.

2. MATERIALS AND METHODS 2.1. Virus origin

A dairy herd was regularly checked serologically between 2000−2004 during a study on BVD epidemiology in Thai-land [7]. All collected serum samples were tested for antibodies to BVDV at the Fac-ulty of Veterinary Medicine and Animal Science, SLU, Sweden, using a commer-cial indirect ELISA (SVANOVA Biotech AB, Uppsala, Sweden). Seronegative sam-ples were further analysed for BVDV anti-gen using a commercial antianti-gen ELISA (Herd Check BVDV Ag/Serum Plus, IDEXX laboratories, Inc., Westbrook, ME,

USA). Due to Swedish legislation, all serum samples were heat inactivated be-fore testing (56 ◦C, 90 min). Between 2003 and 2004, 11 out of 16 previ-ously seronegative animals seroconverted to BVDV without obvious clinical mani-festations, indicating the introduction of a low virulent pestivirus into the herd. By using the antigen ELISA one seronegative viraemic calf was detected.

2.2. Molecular methods

For sequencing, RNA was extracted di-rectly from the antigen positive serum us-ing a GenoM-48 extraction robot (Geno Vision AS, Norway) and the extraction kit MagAttract Virus Mini M48 (Qiagen Gmbh, Germany) according to the standard protocol. A 296-nucleotide (nt) segment of the 5’ NCR of the pestivirus genome was amplified in a one-step RT-PCR, as pre-viously described [14], using the primers OPES 13A and OPES 14A [3]. The gen-erated product was sequenced using the BigDye sequencing kit (BigDye Termi-nator v3.1 Cycle Sequencing Kit, Applied Biosystems, Warrington, UK) according to the instructions of the manufacturer, us-ing the same primers. A 183-nt fragment of the 5’ NCR, corresponding to position 160-341 of BVDV NADL [2], was used for comparative sequence analyses. The nucleotide sequences were assembled and proofread using the SeqMan II and Edit-Seq programmes in the DNASTAR pro-gramme package (DNASTAR Inc., Madi-son, WI, USA), then aligned and compared by the Clustal W method of the Me-gAlign programme from the same pack-age. Additional sequences representative of each known species within the genus

Pestivirus, including the tentative

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using neighbour-joining [12], estimating evolutionary distances with the Kimura 2-parameter method [8]. The robustness of the method was evaluated by bootstrap resampling [4] with 1000 replicates. All phylogenetic analyses were performed us-ing PAUP*, version 4.0 b10 [15].

2.3. Virus neutralisation

From the herd where the viraemic calf was detected, we selected serum sam-ples (collected in 2004) from all animals that seroconverted between 2003 and 2004 (n = 11), based on the results from the indirect ELISA. For comparison, we in-cluded randomly selected samples (n= 22) collected in 2001 from seropositive cat-tle in this and eight previously described herds in the region [7], and a serum sample (VD050916) used as a positive control for serological diagnosis within the Swedish BVD control programme, and that con-sists of pooled sera from a large number of BVDV-antibody positive Swedish cattle. The virus neutralisation (VN) capacity of all sera was tested against representatives from pestivirus species BVDV-1, BVDV-2, and D32/00_‘HoBi’, and those collected in 2004, together with the Swedish pooled serum, also against BDV. Two-fold dilu-tions of all sera were tested in duplicates from dilution 1:8. A fixed dose of virus (100 TCID50) was mixed with 50µL sera and incubated in microtitre plates for 1 h at 37 ◦C with 5% CO2. The cells (50µL bovine turbinate cells, 3× 105_{/mL) were} added and the cultures were incubated for 3 days at 37 ◦C with 5% CO2. The plates were fixed and dried, then stained with immunoperoxidase as described by Meyling [10]. A VN antibody titre 8 was considered a positive result, and the end-point titre was determined by the dilution of serum that showed complete VN activ-ity in both duplicates.

3. RESULTS

3.1. Phylogenetic analysis

The phylogenetic analysis clearly identified six major branches, supported by high bootstrap values, and demon-strated that the detected virus (named Th/04_KhonKaen1_{) was closely related} to D32/00_‘HoBi’ (Fig. 1). The pairwise evolutionary distance within the studied fragment between Th/04_KhonKaen and D32/00_‘HoBi’ was estimated at 0.08. The ranges of pairwise distances between Th/04_KhonKaen and the other species within the genus Pestivirus, including the pestivirus of giraﬀe, were 0.28-0.43 (Tab. I).

3.2. Virus neutralisation

All sera collected in 2004 had very high VN titres against D32/00_‘HoBi’; in most cases markedly higher than against BVDV-1, BVDV-2 or BDV. Consequently, these results supported the results from the phylogenetic analysis, and clearly demon-strated that a ‘HoBi’-like virus had been introduced to, and was now circulating in the herd. The Swedish serum and Thai sera collected in 2001, however, had high VN titres against NADL, but only low or moderate titres against the other pes-tiviruses (Tabs. II and III, respectively).

4. DISCUSSION

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Figure 1. Unrooted phylogram generated from a 183-nucleotide fragment of the 5’ NCR

se-quences of selected representatives of each known species within the genus Pestivirus, including the tentative pestivirus of giraﬀe, the D32/00_‘HoBi’ and Th/04_KhonKaen. The sequences of the virus strains NADL, Osloss, US890, Giraﬀe H138, Brescia, Alfort187, BDV137/4, Moredun, Reindeer-1 and D32/00_‘HoBi’ were obtained from the GenBank (accession nos. NC_001461, M96687, Z79772, AB040131, AF091661, X87939, U65052, U65022, NC_003677, AY489116). Strain CVL-178003/GB-1987 was kindly provided by T. Drew (VLA, UK) and included in the alignment. Values from bootstrap resampling with 1000 replicates are indicated for nodes with val-ues above 60%.

our serological studies clearly revealed the genetic and antigenic relationship between the virus that was circulating in the herd and D32/00_‘HoBi’, and supported the proposition of this group of viruses as rep-resentatives of a novel pestivirus species. The isolation of D32/00_‘HoBi’ from a batch of FCS from Brazil implied a risk

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Table I. Pairwise sequence distances based on a 183-nucleotide fragment of the 5’ NCR sequences

of selected representatives of each known species within the genus Pestivirus, including the tentative pestivirus of giraﬀe, the D32/00_‘HoBi’ and Th/04_KhonKaen.

Strain Pairwise sequence distance

1 2 3 4 5 6 7 8 9 10 11 12 1. D32/00_HoBi – 2. Th/04_KhonKaen 0.08 – 3. BVDV-1 (NADL) 0.35 0.36 – 4. BVDV-1 (Osloss) 0.34 0.32 0.11 – 5. BVDV-2 (890) 0.25 0.31 0.27 0.28 – 6. BVDV-2 (178003) 0.24 0.30 0.28 0.27 0.05 – 7. BDV (Moredun) 0.45 0.43 0.40 0.44 0.42 0.41 – 8. BDV (137/4) 0.46 0.43 0.37 0.42 0.39 0.40 0.08 – 9. Reindeer-1 0.39 0.38 0.34 0.38 0.36 0.38 0.12 0.11 – 10. CSFV (Alfort187) 0.40 0.43 0.46 0.46 0.49 0.48 0.33 0.39 0.32 – 11. CSFV (Brescia) 0.37 0.37 0.34 0.33 0.41 0.39 0.24 0.29 0.22 0.11 – 12. Giraﬀe 0.26 0.28 0.38 0.37 0.32 0.27 0.41 0.33 0.34 0.40 0.31 –

Table II. Virus neutralisation titres of 11 sera

(1–11) collected from cattle in a dairy herd in Thailand, 2004, and of one pooled serum (12) from Swedish cattle. Antibodies were de-tected against representative strains of pes-tivirus species BVDV-1, BVDV-2, BDV and the atypical pestivirus strain D32/00_‘HoBi’.

Sera Pestivirus strain

BVDV-1a _BVDV-2b _BDVc _HoBid 1 16 1024 128 8192 2 32 < 8 64 8192 3 64 128 16 2048 4 64 512 8 8192 5 64 2048 16 4096 6 128 64 32 2048 7 256 64 64 1024 8 256 64 64 2048 9 256 256 64 2048 10 256 256 128 4096 11 512 64 128 2048 12e ₁₀₂₄ ₄ ₆₄ ₁₂₈ a_{Strain NADL.}

b_{Strain CVL-178003}_{/GB-1987, kindly provided by}

T. Drew, VLA, Addlestone, UK.

c_{Strain 137}_{/4, kindly provided by T. Drew, VLA,}

Addlestone, UK.

d _{Kindly provided by M. Beer, Friedrich-Loe}

ﬄer-Institut, Greifswald-Insel Reims, Germany.

e_{Pooled serum (VD050916) used as a positive}

con-trol within the Swedish BVD concon-trol programme.

contaminated vaccines produced with in-fected batches of FCS, or imported se-men or embryos, and (b) that it may already be spread in cattle populations. The fact that pestiviruses are serologi-cally cross-reactive and, at the same time, antigenically and genetically diverse [11], makes this suggestion feasible. It has been observed that, due to the genetic diﬀer-ences, the established pan-pestivirus PCR, using primer-pair 324/326 [16] might fail to detect ‘HoBi’-like viruses [13]. More-over, the serological cross-reactivity com-plicates diﬀerentiation through serologi-cal assays, as illustrated by the results of our VN studies. Had we not included D32/00_‘HoBi’ in the panel of viruses, the interpretation would have been that the herd was exposed to a mixed infection with BVDV-1 and -2.

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Table III. Virus neutralisation titres of sera

from 22 cattle, collected in 9 dairy herds in Thailand, 2001. Antibodies were detected against representative strains of pestivirus species BVDV-1, BVDV-2, and the atypical pestivirus strain D32/00_‘HoBi’.

Sera Pestivirus strain

BVDV-1a _BVDV-2b _HoBic 1 4096 512 256 2 4096 512 256 3 4096 256 64 4 512 64 32 5 2048 32 16 6 2048 128 256 7 4096 64 32 8 512 512 16 9 512 64 32 10 4096 64 8 11 4096 256 64 12 1024 64 128 13 4096 128 512 14 512 16 64 15 4096 1024 128 16 4096 64 128 17 512 < 8 < 8 18 1024 256 512 19 4096 512 512 20 4096 64 512 21 4096 8 256 22 4096 512 256 a_{Strain NADL.}

b _{Strain CVL-178003}_{/GB-1987, kindly provided}

by T. Drew, VLA, Addlestone, UK.

c_{Kindly provided by M. Beer, Friedrich-Loe}

ﬄer-Institut, Greifswald-Insel Reims, Germany.

of the BVD eradication programme [9], and with > 98% of the cattle population being free from and, thus, susceptible to BVDV, an introduction of atypical BVDV strains must be detected rapidly. If not, it would be a serious drawback that could have important economic consequences for the cattle industry. Therefore, we suggest

that representatives of this group of viruses should be included in serological screening assays of cattle used in BVDV control pro-grammes, and that live vaccines and other biological products should be tested for contamination with atypical pestiviruses to ensure good biosafety.

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